Chromatography is a well-characterized technique for isolating analytes, including often complex mixtures made up of a number of natural products. Such isolation is important to identify and further characterize compounds or impurities, and for use in a range of applications including assays to identify relevant candidate compounds based on activity, bioactivity or structure, and downstream applications related to pharmaceutical manufacture and chemical products. An important chromatographic technique is liquid-liquid chromatography that employs a two-phase immiscible solvent system for separating one or more analytes present as solutes in the system. For example, centrifugal partition chromatography (CPC) and countercurrent chromatography (CCC) are high-resolution separation techniques that can fractionate complex mixtures under mild conditions without adversely impacting the mixture constituents. The fractionation is based on the relative analyte solubility in the two immiscible phases.
The number of patents related to CCC provides an indication of the importance of this method in separating compounds (see, e.g., U.S. Pat. Nos. 4,051,025; 4,228,009; 5,217,608; 5,332,504; 5,354,473; 5,449,461; 5,770,083; 6,503,398; EP 0808455). Although there are many different CCC operating modes known in the art, these techniques all suffer from a common defect related to visualization of the data obtained from the instrument, commonly referred to as a chromatogram. These chromatograms are important because they provide a means for visually and quantitatively identifying the analytes detected by the instrument, and provide an indication as to the potential separation or “purity” of analytes as well as an assessment of the suitability of the solvent system to separate an analyte of interest. For example, if two analytes elute at a similar time, the separated compound will likely be a mixture of two different analytes. This presents a problem for applications requiring an isolated compound of high purity or that depend on the full resolution of analytes. Accordingly, it is important that the proper instrument and system related thereto be selected to provide maximum separation of the constituents that are in the mixture.
This selection is made difficult by the fact that CCC and CPC display conventional chromatograms, where it is impossible to make a meaningful comparison between different instruments, even when identical solvent systems and analytes are provided. The difficulty arises because conventional chromatograms are provided with instrument-dependent parameters plotted on the x-axis such as volume (see, e.g., Friesen and Pauli, J. Liq. Chrom & Related Tech. 28:2777-2806, 2005) or time (see, e.g., U.S. Pat. No. 5,770,083). Accordingly, it is not possible to compare the experimental results from one instrument with another. Further, it is difficult to verify whether an instrument is performing adequately or whether there are any calibration issues.
One means for facilitating instrument comparison is by providing chromatograms where instrument-independent parameters are plotted, such as by plotting a distribution constant (e.g., partition coefficient, partition constant, distribution coefficient, or any other nomenclature reflecting distribution between the immiscible phases), K, rather than a time or volume. An example of such a plot is provided in Friesen and Pauli (Anal Chem. (2007) 79(6):2320-4), where K is plotted on the x-axis and compared against conventional chromatograms.
One disadvantage of such a K-plot (and for plots using conventional parameters such as volume or time) relates to the inability of such plots to encompass all possible analytes, such as a plurality of analytes from a mixture having some analytes with a K-value that is about zero and with a value corresponding to infinity. Such a situation simply reflects the situation where an analyte is confined (e.g., dissolves) completely in only one of the two phases, corresponding to a K-value that is zero or infinity, or approaches one of these values. In addition, as the K-value increases, especially for those K values that are large, the peak width tends to increase. This increase in width can be misinterpreted as a decrease in resolution.
Although partition coefficients are known in the art of CCC (e.g., see U.S. Pat. No. 5,354,473), the methods and techniques presented herein provide a means for standardizing output results from any kind of liquid-liquid chromatographic instrument. Such standardization is valuable in a number of applications ranging from upstream instrument manufacture to downstream user experimental comparison, instrument calibration, solvent selection, and assessment of instrument performance. The methods disclosed herein provide improved means for assessing CCC and partition chromatography, resulting in increased reliability and confidence in the data obtained by such instruments, and can be integrated with any number of a wide range of existing and future-arising CCC and CPC instruments.